AbstractForward genetic screens represent powerful, unbiased approaches to uncover novel components in any biological process. Such screens suffer from a major bottleneck, however, namely the cloning of corresponding genes causing the phenotypic variation. Reverse genetic screens have been employed as a way to circumvent this issue, but can often be limited in scope. Here we demonstrate an innovative approach to gene discovery. Using C. elegans as a model system, we used a whole-genome sequenced multi-mutation library, from the Million Mutation Project, together with the Sequence Kernel Association Test (SKAT), to rapidly screen for and identify genes associated with a phenotype of interest, namely defects in dye-filling of ciliated sensory neurons. Such anomalies in dye-filling are often associated with the disruption of cilia, organelles which in humans are implicated in sensory physiology (including vision, smell and hearing), development and disease. Beyond identifying several well characterised dye-filling genes, our approach uncovered three genes not previously linked to ciliated sensory neuron development or function. From these putative novel dye-filling genes, we confirmed the involvement of BGNT–1.1 in ciliated sensory neuron function and morphogenesis. BGNT–1.1 functions at the trans-Golgi network of sheath cells (glia) to influence dye-filling and cilium length, in a cell non-autonomous manner. Notably, BGNT–1.1 is the orthologue of human B3GNT1/B4GAT1, a glycosyltransferase associated with Walker-Warburg syndrome (WWS). WWS is a multigenic disorder characterised by muscular dystrophy as well as brain and eye anomalies. Together, our work unveils an effective and innovative approach to gene discovery, and provides the first evidence that B3GNT1-associated Walker-Warburg syndrome may be considered a ciliopathy.Author SummaryModel organisms are useful tools for uncovering new genes involved in a biological process via genetic screens. Such an approach is powerful, but suffers from drawbacks that can slow down gene discovery. In forward genetics screens, difficult-to-map phenotypes present daunting challenges, and whole-genome coverage can be equally challenging for reverse genetic screens where typically only a single gene’s function is assayed per strain. Here, we show a different approach which includes positive aspects of forward (high-coverage, randomly-induced mutations) and reverse genetics (prior knowledge of gene disruption) to accelerate gene discovery. We paired a whole-genome sequenced multi-mutation C. elegans library with a rare-variant associated test to rapidly identify genes associated with a phenotype of interest: defects in sensory neurons bearing sensory organelles called cilia, via a simple dye-filling assay to probe the form and function of these cells. We found two well characterised dye-filling genes and three genes, not previously linked to ciliated sensory neuron development or function, that were associated with dye-filling defects. We reveal that disruption of one of these (BGNT–1.1), whose human orthologue is associated with Walker-Warburg syndrome, results in abrogated uptake of dye and cilia length defects. We believe that our novel approach is useful for any organism with a small genome that can be quickly sequenced and where many mutant strains can be easily isolated and phenotyped, such as Drosophila and Arabidopsis.
Repression of an activity-dependent autocrine insulin signal is required for sensory neuron development in C. elegans
